This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Estrogens affect a large number of physiological functions and disease states. In addition to endogenous hormones, humans are exposed to a wide variety of estrogenic compounds in the diet, environment, and pharmaceuticals. These non-classical ligands for the estrogen receptor (ER) can have major effects on human health. Aim 1: Efficient methods of testing for estrogenic activity are important for drug development, environmental testing, and research on receptor functions and mechanisms. Yeast-based assays using exogenously expressed ERs and reporter genes are inexpensive and widely used, but have the defect that the response to some SERMS (selective estrogen receptor modulators) and phytoestrogens is very different from that seen in mammalian cells. This project will develop and test a reporter system in the nematode C. elegans, which is easily grown in culture. It is hypothesized that the closer evolutionary relationship of humans and C. elegans will enable this system to produce more relevant results than yeast-based assays, but retain the advantages of simplicity and low cost. Aim 2: Although there is substantial human exposure to non-classical ER ligands, their modes of action are incompletely understood. SERMs such as tamoxifen often have biphasic dose-response curves, with ER agonism at low concentrations and antagonism at high concentrations. It has been proposed that in addition to occupying the hormone-binding pocket of ER, these compounds bind (with lower affinity) to a 2nd site that mediates antagonism. Some phytoestrogens have a similar biphasic effect, suggesting that they are natural ligands for this site. This project will determine if phytoestrogens and ANGELS (activators of non-genotropic estrogen-like signaling, a new class of ER-targeted drugs) compete with SERMS for binding to the 2nd site, identify and ablate the site via targeted mutagenesis, and characterize the significance of the site in ER function by reporter gene assays, microarray analysis and real-time PCR.